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Abstract

The biggest challenge in the field of gene therapy is how to effectively deliver target
genes to special cells. This study aimed to develop a new type of poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable
of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposome-based
gene carrier, such as the cytotoxicity induced by excess positive charge, as well
as the aggregation on the cell surface. The PLGA-based nanoparticles presented in
this study were synthesized by emulsion evaporation method and characterized by transmission
electron microscopy, dynamic light scattering, and energy dispersive spectroscopy.
The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60
nm at the optimal charge ratio. Without observable aggregation, the nanoparticles
showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier
for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency
and lower cytotoxicity in HepG2 cells compared to the PEI/DNA complex. The N/P ratio
(ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex
displays the best property among various N/P proportions, yielding similar transfection
efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows
better serum compatibility than commercial liposome. PLGA nanocomplexes obviously
accumulate in tumor cells after transfection, which indicate that the complexes contribute
to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a
by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI
nanoparticles are promising non-viral vectors for gene delivery.

Introduction

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNAs that regulate gene
expression at the post-transcriptional level. They involve in various cellular mechanisms
including development, differentiation, proliferation, and apoptosis. The pivotal
roles of these miRNAs in human cancers have been discovered [1,2], and the therapeutic applications of miRNA have been developed using various viral
vectors [3,4].

However, the disadvantages of viral vectors limited their application in gene delivery,
such as immunogenic/inflammatory responses, low loading capacity, large scale manufacturing,
and quality control [5]. Consequently, more attention have been paid on non-viral gene delivery vectors in
recent years, such as liposomes (lipoplexes), polycationic polymers (polyplexes),
and organic or inorganic nanoparticles (nanoplexes) [6]. To enhance gene delivery effect, various cationic complexes have been developed
for delivering plasmid DNA, antisense, or siRNA into cells [7-9]. Poly(D,L-lactide-co-glycolide) (PLGA) were extensively assessed for their ability of delivering
variety of therapeutic agents [10-12]. PLGA nanoparticles were shown to escape from the endo-lysosomal compartment to the
cytoplasmic compartment and release their contents over extended periods of time [13]. These features rendered PLGA nanoparticles as potential tool for gene delivery efficiently.

Polyethylenimine (PEI) is water-soluble, linear, or branched polymers with a protonable
amino group [14,15]. Due to their high cationic charge density at physiological pH, PEIs are able to
form non-covalent complexes with DNA, siRNA, and antisense oligodeoxynucleotide. Therefore,
PEIs hold a prominent position among the polycationic polymers used for gene delivery
[16-18]. The intracellular release of PEI/nucleic acids complexes from endosomes is considered
as relying on the protonation of amines in the PEI molecule, which leading to osmotic
swelling and subsequent burst of the endosomes. Moreover, PEIs also facilitate nucleic
acid entry into the nucleus [19,20]. However, it has been reported that long PEI chains are highly effective in gene
transfection, but more cytotoxic [14,21,22].

In order to overcome these hurdles in gene therapy and improve gene delivery efficiency,
we developed novel non-liposome-based cationic polymers which are composed of PLGA
as the core and cationic PEI as the shell. The biodegradable PLGA nanoparticles, modified
with a polyplexed PEI coating, were tested by loading the expression vector (pDNA)
of miR-26a, which is capable of inducing cell cycle arrest in HepG2 cells. In this
study, nanoparticles of controlled size and persistent shape have been obtained by
an emulsion evaporation method and characterized by transmission electron microscopy
(TEM), dynamic light scattering (DLS), and energy dispersive spectroscopy (EDS). The
nanoparticles have been determined by their physicochemical and biological properties.
The formulated nanoparticles enhance cellular uptake of miRNA, pronounce upregulation
of miR-26a, induce cell cycle arrest, and improve gene expression activity compared
with PEI and commercial liposome. Furthermore, these particles can be easily fabricated
and have a high transfection efficiency and low cell toxicity. Our results suggest
a new approach for miRNA delivering by PLGA/PEI nanoparticles in gene therapy.

PLGA/PEI nanosphere synthesis

PLGA nanospheres were obtained by using water-in-oil-in-water solvent evaporation
technique as described previously [24]. Briefly, 150 mg of PLGA polymer was dissolved in 1.5 ml of dichloromethane to yield
a 10% (w/v) polymer solution. After 3 ml of a 7% (w/v) aqueous solution of PVA was added to the organic phase and emulsified at 10,000
× g using a homogenizer for 5 min. The resulting double emulsion was then poured into
50 ml of a 1% PVA solution and emulsified for 15 min. This resulted in the formation
of a water/oil/water emulsion that was stirred for at least 12 h at room temperature,
allowing the methylene chloride to evaporate. The resulting microspheres were washed
twice in deionized water by centrifugation at 16,000 × g and freeze-dried.

The zeta-potential (surface charge) of the polymers and polyplexes was determined
at 25°C with a scattering angle of 90° using potential measurement analyzer (90 PLus,
Brookhaven, Holtsville, NY, USA). Samples were prepared in PBS and diluted with deionized
water to ensure that the measurements be performed under conditions of low ionic strength
where the surface charge of the particles can be measured accurately.

Measurement of the interactions between miRNA and nanoparticles

Complexes were formed by diluting miRNA expression vector (pDNA) and the different
amount of nanoparticles separately with 0.9% NaCl (pH 7.4). The nanoparticles at different
concentrations were added to 1 μg green fluorescent protein(pPG-eGFP-miR) (GFP)-encoded
miRNA vector solution, vortexed immediately at room temperature, and allowed to stand
for 30 min to form PLGA/PEI/pDNA complexes. Then, the complexes were submitted to
electrophoresis in 1%/TAE agarose gel at 90 V for 60 min. Images were acquired using
a PeiQing gel imaging system (PeiQing, Shanghai City, China). GelRed (Biotium, Hayward,
CA, USA), an ultra sensitive nucleic acid dye, was used to examine the interactions
of DNA with the nanocomplex to determine optimal N/P ratio of the nanocomplex [25].

Cytotoxicity studies

HepG2 (human hepatocellular carcinoma cell) cells were cultured in DMEM supplemented
with 10% FBS, streptomycin at 100 μg/mL, and penicillin at 100 U/mL. Cells were maintained
at 37°C in humidified and 5% CO2 incubator. The cytotoxicity of PLGA/PEI nanoparticles, complexed with or without pDNA,
was determined in a separate set of experiments using MTT assay to detect changes
in cell viability after an incubation time of 24 h. Cells were seeded in 24-well plates
at an initial density of 2 × 104 cells/well for HepG2 in 0.5 mL of growth medium and incubated for 24 h prior to the
addition of PLGA/PEI and PLGA/PEI/DNA at different N/P ratio. Untreated cells were
taken as control with 100% viability. Triton X-100 1% (SPI Supplies, West Chester,
PA, USA) was used as positive control of cytotoxicity.

In vitro gene transfection and quantification study

Before GFP transfection assay, cells were seeded in 24-well plates at a density dependent
of the cell line in DMEM with 10% FBS. When the cells were at 50% to 70% confluence,
the medium in each well was replaced with fresh normal medium or medium containing
naked pGFP, lipo2000, or PLGA/PEI/DNA complex under standard incubator conditions.
After 48 h, cells harboring an expressing integrant were viewed by fluorescence microscopy
based on GFP.

The analysis of transfection efficiency was performed using a flow cytometer (BD Biosciences,
Mountain View, CA, USA). Cells were first washed with PBS and detached with 0.2 mL
of 0.25% trypsin. Growth medium was then added, and the cells suspension was centrifuged
at 1,000 rpm for 5 min. Two further cell-washing cycles of resuspension and centrifugation
was carried out in PBS before fixation in 0.4 mL of 75% ethanol. The percentage of
cells expressing GFP was then determined from 10,000 events and reported as a mean
± standard deviation (SD) of three samples.

miRNA expression and cell cycle study

Gene-specific primers and reverse transcriptase were used to convert mature miRNA
to cDNA [26], DNase-treated total RNA (20 μl of total volume) was incubated with 1 μl of 10 mM
reverse transcription primers listed in Table 1. The reaction was heated to 80°C for 5 min to denature the RNA and then cooled to
room temperature quickly, after that the remaining reagents (5 × buffer, primescript
RTase (TaKaRa, Dalian, China), dNTPs, DTT, and RNase inhibitor(SunshineBio, Nanjing,
China) were added as specified according to the manufacturer's protocol. The reaction
proceeded for 45 min at 42°C followed by 5 min incubation at 85°C to inactivate the
reverse transcriptase. cDNA may be stored at -20°C or -80°C.

Then, real-time quantitative polymerase chain reaction (PCR) was performed to evaluate
miR-26a expression in HepG2 cells after 3 days transfection using standard protocols
on an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster
City, CA, USA). Briefly, 1.25 μl of cDNA was added to 10 μl of the 2 × SYBR green
PCR master mix (TaKaRa, Dalian, China), 200 nM of each primer and water to 20 μl.
The reactions were amplified for 15 s at 95°C and 1 min at 60°C for 40 cycles. The
thermal denaturation protocol was run at the end of the PCR to determine the number
of the products that were present in the reaction. Reactions are typically run in
triple. The cycle number at which the reaction crossed an arbitrarily placed threshold
(Ct) was determined for each gene and the relative amount of each miRNA. Target gene
expression was normalized to the expression of the housekeeping gene U6 for each sample.
Data were analyzed using the method [27].

Cell cycle profiles of nanoparticles transfected HepG2 cells were performed using
a FACS Calibur and with CellQuest™ software (BD Biosciences, Mountain View, CA, USA).
HepG2 cells (5 × 104 per well) were seeded into 24-well plates. Cells were treated with different formulations
at a concentration of 1 μg miRNA in serum containing medium at 37°C for 72 h. Cells
were washed once with PBS and then fixed in 75% ethanol and, after fixation, stained
with PI according to manufacturer's instructions.

Statistical analysis

All statistical analyses were performed by Student's t test. Data were expressed as means ± SD. Results were considered statistically significant
when the p value was less than 0.05.

Results and discussions

Particle size and surface morphology of the nanoparticles

Particle size and zeta potential have been demonstrated to play important roles in
determining the level of cellular and tissue uptake; researchers have also conducted
to investigate the size effect on the gene delivery efficiency. PLGA nanoparticles
with smaller size below 100 nm have been proved to gain higher gene transfection efficiency
than those of 200 nm [28,29].

In this study, PLGA-based nanoparticle was developed and characterized using TEM,
DLS, and EDS. Figure 1 showed different characteristics of PLGA nanoparticles with PEI or PEI/pDNA. The
diameter of the nanoparticles/DNA complexes ranged from 45 to 70 nm at the determined
nanoparticles/DNA N/P ratios. Slight size changes were observed following coating
of the biodegradable PLGA nanoparticles with PEI or PEI/pDNA. The mean diameter of
the sole PLGA nanoparticles was approximately 50 nm, whereas the sizes of complexed
PLGA nanoparticles containing PEI or PEI/pDNA are increased to about 57 or 60 nm,
respectively. These results are consistent with our DLS observations and indicate
that the PLGA nanoparticle size may be increased with PEI or polyplexed PEI/pDNA coating.

Zeta potential is an indicator of the surface charge of nanoparticles/DNA complexes.
The positive charges on the surface of the complexes can help the nanoparticles bind
tightly to the negatively charged cellular membrane, therefore facilitating their
entry into the cells by endocytosis. In this study, zeta potential values of the PLGA
nanoparticles and PLGA/PEI nanoparticle and PLGA/PEI/pDNA complexes (at various N/P
ratios ranging from 1 to 10) were measured. The values of the PLGA nanoparticles,
PLGA/PEI nanoparticles, and PLGA/PEI/pDNA complexes are -21.4, 29.4, and 23.7 mV,
respectively. As shown in Table 2, the pure PLGA nanoparticles represent negative potential due to the existence of
carboxyl. PEI is postulated on the surface of PLGA nanoparticles due to the electrostatic
interaction of cationic PEI molecules with anionic PLGA polymers. Introduction of
PEI increase significantly the zeta potential of the nanoparticles. Again, pDNA are
adsorbed on the surface of PLGA/PEI nanoparticles due to the electrostatic interaction
of negatively charged pDNA with positively charged PLGA/PEI nanoparticles. The zeta
potential of the complexes increase in parallel with the nanoparticles/pDNA N/P ratio,
ranging from -21.4 to +23.7 mV, which results in a good affinity to cell surface.

EDS is capable of providing both qualitative and quantitative information about the
presence of different elements in nanoparticles. In this study, the surface modification
of PLGA nanospheres with PEI or PEI/pDNA was confirmed by EDS. Because PEI contains
the nitrogen element and pDNA contains phosphorus element, but PLGA contains neither
of them, the nitrogen that is detected in PLGA/PEI nanoparticles is an evidence for
the existence of PEI on the PLGA nanoparticles surface (Table 2). By detecting of phosphorus PLGA/PEI/pDNA nanocomplex, we have shown that pDNA has
been successfully adsorbed on the PLGA/PEI surface. From the combination of the aforementioned
data, it can be inferred that the PEI or pDNA is completely complexed with the nanoparticles
by ionic binding.

Characterization of nanoparticles/pDNA complexes

To explore complex formation of pDNA and PLGA/PEI nanoparticles, PLGA/PEI nanoparticles
were mixed with pDNA at various ratios. Complex formation was assessed by a gel retardation
assay. The pDNA was vortexed with the nanoparticles in nuclease-free water at different
N/P ratios. As shown in Figure 2, the nanoparticles are able to fully retard the mobility of DNA in agarose gel when
the N/P ratio is 10:1 or higher than 10:1. While N/P is lower than 10:1, the retardation
is not full. DNA bands are visible in N/P complexes in lanes 2 to 4 (Figure 2), indicating the presence of free DNA in nanocomplexes of 1 and 4 N/P ratios. This
result is comparable with the band observed in lane 1, which contain only free plasmid.
Few free DNA bands was observed in subsequent lanes of N/P ratios 6 and 10, indicating
complete complexation of all free plasmid. The results indicate that the nanocomplexes
can be easily prepared by simply mixing cationic polymer and DNA solution, and the
results also showed that the optimal N/P is 6:1.

In vitro gene expression assay

It is clear that gene delivery is dependent on DNA/vector uptake efficiency. Fluorescent
proteins, such as GFP, are usually used to label non-viral vectors for measuring the
uptake efficiency. In this study, nanoparticle/pGFP-miR-26a complexes were employed
for the assessment of the transfection efficiency in HepG2 cells. Firstly, HepG2 cells
were transfected by 2 μg of pDNA complexed with 100 μl polymer at different nanoparticles/pDNA
N/P ratios. The nanocomplexes were further evaluated for their transfection efficiency
in cells by screening the GFP signals with flow cytometry. The results showed that
transfection efficiency reached the optimum at nanoparticles/DNA N/P ratio 6 and further
decreased till the N/P ratio reached 9 (Figure 3E). It is suggested that insufficient surface potential of complexes at low N/P ratios
resulted in lower GFP expression, whereas at high N/P ratios, induction of complexes
undoubtedly result in cytotoxicity because of the excessive PEI in the nanocomplex.

Next, transfection efficiency of the nanoparticles was compared to other different
vehicle. As shown in Figure 3, GFP expression is hardly detected when transfection was mediated by naked pDNA,
which was used as negative control. The results have demonstrated that the naked GFP
is difficult to be directly internalized by the cells. In contrast, GFP-contained
nanoparticles facilitate the cell endocytosis. Figure 3E also showed that transfection efficiency of nanoparticles is comparable to the commercial
liposomes, and obviously better than the sole PEI particles.

Furthermore, PLGA/PEI nanoparticles display the transfection efficiency in a dose-dependent
manner (Figure 3F). The uptake efficiency of nanoparticles by cells, assayed on 48 h after incubation,
was higher at lower nanoparticle concentration. However, the transfection efficiency
deceased when the nanoparticle concentration is further increasing due to the potential
cytotoxicity.

Furthermore, we studied the stability of nanoparticles/pDNA in serum. Results suggested
that transfection efficiency of the nanocomplexes is higher than lipo2000 in the presence
of serum (data not shown). Meanwhile, the GFP expression at the 48 h of incubation
was significantly higher than at 24 h. While the nanoparticle/DNA in the medium was
washed away at 24 h after transfection, after another 24 h, GFP signals were not obviously
enhanced (data not shown). The data indicate that the increasing GFP expression results
from the sustained nanoparticle uptake effect of the transfected cells. It was further
proved that the nanoparticles have good biocompatibility in serum.

In vitro cytotoxicity

MTT assay has been widely used for cell proliferation and biochemical toxicity testing.
In this study, MTT assay was used to investigate the cytotoxicity of nanoparticles/pDNA
complexes on HepG2 cells. Figure 4 showed that the cytotoxicity of PLGA/PEI (N/P 6) nanoparticles is remarkably lower
on HepG2 cells, compared with the pure PEI, and close to commercial liposome. Indeed,
PLGA/PEI/pDNA showed above 90% cell viability at N/P 6. By contrast, cell viability
dropped down about 54% and 86% in the presence of PEI and liposome, respectively;
there is little effect on cell viability observed with or without DNA, and this confirmed
that High Molecular Weight (HMW) PEI aggregates on the cell surface, inducing lysosomal
breakdown and mitochondrial damage, thereby affecting cell viability [30]. Nanoparticles of PLGA as core, coated with PEI, presented in this study, effectively
improve the stability of nanocomposites and avoid the release of toxic free PEI in
the cells after delivery of the miRNA vector.

Figure 4.Viability of HepG2 cells 48 h posttreatment with one of above transfection reagents. Values are the mean average ± SD of 3 wells applied with the same reagents.

miR-26 expression and cell cycle induction

To examine the biological activities of miR-26a delivered by nanoparticles/pDNA in
HepG2 cells, the expression levels of miR-26a were detected by qRT-PCR in transfected
HepG2 cells. Forty-eight hours after transfection, HepG2 cells were harvested and
total RNA was extracted for monitoring miRNA expression. Real-time quantitative RT-PCR
results show that the miR-26a level in the transfected cells is increased by 7.73-folds
compared with untransfected cells (p < 0.05) (Figure 5). In contrast, the expression level of miR-26a shows no obvious change in cells transfected
with negative control (miR-NC) and naked pDNA (p > 0.05).

The miR-26a-containing PLGA-based nanocomposites significantly increase the expression
level of miR-26a and inhibit the cell cycle progression by induction of G1 phase arrested
in transfected HepG2 cells, whereas the effect in control groups (miRNA negative control
transfected cells) were not detectable. Figure 6C indicates that cell populations with enforced miR-26a expression were characterized
by significantly increased numbers of cells arrested in G1, which is more than that
of tumor cells treated with miR-NC containing PLGA nanocomplex or untreated control
(Table 3). Kota et al. have demonstrated that upregulation of miR-26a expression results in
the inhibition of cancer cell proliferation, induction of tumor-specific apoptosis,
and enhancement of antitumor activity [3]. In this study, we have developed a PLGA-based nanocomplex and tested its gene delivery
ability on HepG2 cells. Our studies demonstrate that PLGA nanocomplex facilitated
cellular uptake and enhanced gene expression activity.

Researchers have reported that HMW cationic PEI as a transfection reagents showed
higher cytotoxicity [21,22,31,32]. In our studies, the formulation of PLGA-based nanoparticle significantly reduces
the cytotoxicity of the PEI. We have demonstrated that PLGA/PEI nanocomplex shows
higher gene transfection efficiency and better serum compatible than Lipofectamine2000
or PEI. As for the possible reasons, we speculated that the enhanced transfection
effect may be related to the interaction between PLGA and PEI. In the given nanoparticles
mentioned above, PLGA showed a better biocompatibility than the lipids. Obviously,
the amino groups of PEI play an important role in determining the biological characteristics
of the nanocomplexes. To our knowledge, there are few scientific articles describing
solid preformed PEI-based nanoparticles and their cellular applications. Moreover,
in the most of these studies, PEI is complexed through cooperative electrostatic interactions
with other anionic polymers or converted into nanoparticles by introducing ionic and
covalent crosslinkers without any addition of other polymers.

Furthermore, the properties of the nanoparticles we prepared can be easily controlled.
Their size can be tuned by modulating experimental conditions of preparation, and
the surface charge can be adjusted by changing the PEI amount introduced into the
complex. Finally, cytotoxicity studies showed that the PLGA/PEI/DNA complex exhibited
less cytotoxicity than HMW PEI and liposome. In the tested cell line, the transfection
efficiencies mediated by PLGA/PEI increased with the increase of nanoparticles/DNA
NP ratio in the presence or absence of serum.

Conclusion

In this study, we developed a PLGA-based nanoparticles polyplexed with miRNA expression
vector as a potential approach to deliver genes with non-viral vectors. The biodegradable
nanoparticles can efficiently deliver nucleic acid to human hepatocellular carcinoma
cells and enhance the effect of functional miRNA delivered, such as the cell cycle
suppressor miRNA26a. Importantly, it is proved that serum cannot inhibit the transfection
activity of this nanoparticle. These PLGA-based nanoparticles also display an improved
safety profile in comparison with high molecular weight PEIs and liposome because
of the lower cytotoxicity of the polyplex formulations. This study presents an effective
gene delivery vehicle, PLGA-based nanoparticle, which may contribute to the gene therapy
for tumor and other miRNA-related diseases such as diabetic, cardiovascular disease,
and neurodegenerative diseases.

Authors' contributions

GFL designed the experiment, carried out the molecular biologic studies and drafted
the manuscript. YLZ carried out the preparation and characterization of nanoparticles
drafted the manuscript. ZDX conceived of the study, and participated in its design
and coordination. All authors read and approved the final manuscript

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

The authors are grateful for financial support fromthe National Basic Research Program
of China (973 Program: 2007CB936300), NSFC (no. 20875014, no. 30870626), and 2008DFA51180.

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